*2.10. Antimicrobial Tests*

2.10.1. MICs and MBCs Determination of TO@NZ Hybrid Nanostructure against LAB and Pathogen Bacteria

The MICs and MBCs of TO@NZ against different bacteria are presented in Table 4.

**Table 4.** Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of TO@NZ hybrid nanostructure against lactic acid bacteria and pathogens (% *w*/*v*).


TO@NZ hybrid nanostructure exerted antimicrobial activity against all tested bacteria for at least 2 days, although its effectiveness was dependent on the targeted strain. It was observed that both the lactic acid bacteria were inhibited at 0.025% concentration, which was the lowest MIC compared with that of the pathogenic strains. However, the MICs measured for the pathogenic strains were two times higher (0.05%) for *S. aureus* and 20 times higher (0.1) for *L. monocytogenes* and *Enterococcus faecalis* strains. These results indicate that the hybrid nanostructure can inactivate LAB and presumptive pathogenic microorganisms associated with spoilage and the safety of dairy products. Similar results have been reported [51] for the antimicrobial activity of thyme essential oil in Coahlo fresh cheese, where the MIC of thyme oil on pathogenic *S. aureus* and *L. monocytogenes* was two times higher (2.5 µL/mL) than that (1.25 µL/mL) for starters *L. lactis* ssp. *lactis* and *L. lactis* ssp. *cremoris.* These results suggest that the doses of EO thyme alone and in the form of TO@NZ hybrid nanostructure to control pathogenic bacteria should affect the growth and survival of starter cultures. On the other hand, carvacrol and thymol have been reported as the most inhibitory essential oils against non-starter lactic starter bacteria (NSLAB) with MICs of 0.1% (*w*/*v*) [52]. Reduction of 2-log CFU/mL against *L. buchneri* and

*P. acidilactici* was achieved for thymol 0.1% (*w*/*v*) while this concentration was bactericidal against *L. citrovorum* (>4-log reduction). These results indicate that thymol oil can inactivate the non-starter LAB. According to the common opinion, such bacteria cause spoilage of shelf-stable low-acid dairy products. In particular, antimicrobial activity of thyme oil on milk and dairy products was established by previous studies [53,54].

#### 2.10.2. Antimicrobial Activity of Active Films Application on Cheese against *S. aureus Gels* **2022**, *8*, x FOR PEER REVIEW 15 of 24

The results for the antimicrobial activity of hybrid nanostructured films used for packaging of cottage cheese under 10 ◦C storage temperature are shown in Figure 11.

**Figure 11.** Changes in *Staphylococcus aureus* ATCC1534 counts in cottage cheese samples wrapped with alginate-based edible films (ALG/G/xTO@NZ) with the hybrid nanostructure TO@NZ in different concentrations. **Figure 11.** Changes in *Staphylococcus aureus* ATCC1534 counts in cottage cheese samples wrapped with alginate-based edible films (ALG/G/xTO@NZ) with the hybrid nanostructure TO@NZ in different concentrations.

Given the strong antibacterial activity of the TO@NZ bioactive hybrid nanostructure against the pathogen strains, the prepared ALG/G/TO@NZ active films were studied for the antimicrobial activity against the pathogenic bacteria *S. aureus* ATCC1538 in cottage fresh cheese, as a frequently associated toxinogenic bacteria with fresh or low-ripened cheeses. During 15 days of storage of cheese at an abuse temperature of 10 °C, it was found that *S. staphylococcus* ATCC1538 levels increased in the control uncoated sample and the sample coated with ALG/G film. However, a significant decrease of this bacterium concentration occurred in all cheese samples coated with ALG/G/xTO@NZ films. Furthermore, the antimicrobial activity was significantly related with films containing essential oils (*p* < 0.05). The initial count of *S. aureus* ATCC1538 in the uncoated control sample was 4.25 log10 cfu/g on the 1st day of storage and this value significantly increased (*p* < 0.05) to 4.75 (0.50 log10 cfu/g increase) by the 15th day of storage. However, in the case of the coated sample with the ALG/G film without the TO@NZ hybrid nanostructure, there was a slight decrease (not statically significant) of count to 4.1 log10 (cfu/g), which shows a bacteriostatic activity on *S. aureus* strain*.* The application of ALG/G/5TO@NZ active film on cheese resulted in a significant reduction of about 1-log10 (cfu/g) (*p* < 0.05) against the *S. aureus* ATCC1538 population from the 2nd day of storage, which remained at this level during the whole storage period (15 days), indicating a bacteriostatic activity. However, in the case of ALG/G/10TO@NZ and ALG/G/15TO@NZ active films application on cheese, a sig-Given the strong antibacterial activity of the TO@NZ bioactive hybrid nanostructure against the pathogen strains, the prepared ALG/G/TO@NZ active films were studied for the antimicrobial activity against the pathogenic bacteria *S. aureus* ATCC1538 in cottage fresh cheese, as a frequently associated toxinogenic bacteria with fresh or low-ripened cheeses. During 15 days of storage of cheese at an abuse temperature of 10 ◦C, it was found that *S. staphylococcus* ATCC1538 levels increased in the control uncoated sample and the sample coated with ALG/G film. However, a significant decrease of this bacterium concentration occurred in all cheese samples coated with ALG/G/xTO@NZ films. Furthermore, the antimicrobial activity was significantly related with films containing essential oils (*p* < 0.05). The initial count of *S. aureus* ATCC1538 in the uncoated control sample was 4.25 log<sup>10</sup> cfu/g on the 1st day of storage and this value significantly increased (*p* < 0.05) to 4.75 (0.50 log<sup>10</sup> cfu/g increase) by the 15th day of storage. However, in the case of the coated sample with the ALG/G film without the TO@NZ hybrid nanostructure, there was a slight decrease (not statically significant) of count to 4.1 log<sup>10</sup> (cfu/g), which shows a bacteriostatic activity on *S. aureus* strain. The application of ALG/G/5TO@NZ active film on cheese resulted in a significant reduction of about 1-log<sup>10</sup> (cfu/g) (*p* < 0.05) against the *S. aureus* ATCC1538 population from the 2nd day of storage, which remained at this level during the whole storage period (15 days), indicating a bacteriostatic activity. However, in the case of ALG/G/10TO@NZ and ALG/G/15TO@NZ active films application on cheese, a significant decrease of 2 log<sup>10</sup> (cfu/g) of *S. aureus* population was counted from the

nificant decrease of 2 log10 (cfu/g) of *S. aureus* population was counted from the 2nd day of storage and from the 5th day no counts detected up to 15th day, indicating a strong

(WPIOF) and *S. aureus* inactivation in Kashar cheese [55]. Opposite of the studies in which the essential oils were added directly to foods, the number of studies in which the essential oils were used as a composite of packaging materials are fewer [56]. As the time passes or/and the temperature increases the possibility for food illness to arise increases, given that an *S. aureus* population of 105 cfu/g consists the threshold for alpha-staphylotoxin 2nd day of storage and from the 5th day no counts detected up to 15th day, indicating a strong bactericidal effect of both active films against the pathogen. Similarly, a significant relationship has been detected between the thyme-fortified edible whey isolate-based films (WPIOF) and *S. aureus* inactivation in Kashar cheese [55]. Opposite of the studies in which the essential oils were added directly to foods, the number of studies in which the essential oils were used as a composite of packaging materials are fewer [56]. As the time passes or/and the temperature increases the possibility for food illness to arise increases, given that an *S. aureus* population of 10<sup>5</sup> cfu/g consists the threshold for alpha-staphylotoxin production in cheese [56]. In our study, the cheese was strongly protected from Staphylococcal intoxication even under abusing storage temperature of 10 ◦C. However, we should consider that the application of ALG/G/TO@NZ active films in fermented dairy products (such as cheeses) in doses enough to control pathogenic bacteria could also influence the growth and survival of lactic acid bacteria, which probably affect the post-acidification activity and improve the product's self-life [57].
